The invention relates in general to transfection of cells and to agents which condense nucleic acid.
Cell transfection relies on efficient delivery of DNA to target cells, and expression of the delivered DNA in the nucleus of such cells.
Early experiments on introducing DNA into mammalian cells in vitro utilized DNA in precipitated form with low efficiency of transfection and required selectable marker genes (Wigler et al. (1977) Cell 16:777-85; Graham and Van der Eb (1979) Proc. Natl. Acad. Sci. USA 77:1373-76 and (1973) Virology 52:456)). Since this time molecular biologists have developed many other more efficient techniques for introducing DNA into cells, such as electroporation, complexation with asbestos, polybrene, DEAE, Dextran, liposomes, lipopolyamines, polyornithine, particle bombardment and direct microinjection (reviewed by Kucherlapati and Skoultchi (1984) Crit. Rev. Biochem. 16:349-79; Keown et al. (1990) Methods Enzymol. 185:527). Many of these methods are unsuitable for use clinically since they give highly variable and relatively poor levels of transfection. Another obstacle to the wider use of existing transfection complexes resides in their instability in vivo. It has been shown that particles of a similar size to the transfection complexes of the prior art are rapidly and efficiently removed from the blood by the reticuloendothelial system (Poste and Kirsch, Bio/Technology 1:869 (1984)).
Soluble DNA/polylysine complexes have been generated (Li et al., (1973) Biochem. J. 12:1763) and tagged with asialoglycoprotein to target DNA to hepatocytes in vitro (Wu and Wu, J. Biol. Chem. 262:4429 (1987); U.S. Pat. No. 5,166,320). Lactosylated polylysine (Midoux et al. (1993) Nuc. Acids Res. 21:871-878) and galactosylated histones (Chen et al. (1994) Human Gene Therapy 5:429-435) have been used to target plasmid DNA to cells bearing lectin receptors, and insulin conjugated to polylysine (Rosenkrantz et al. (1992) Exp. Cell Res. 199:323-329) to cells bearing insulin receptors. However, Wagner et al. (supra) have shown that the latter approach is even less efficient than standard methods of transfection, and may therefore be considered unsuitable for pharmaceutical-development. Monoclonal antibodies have been used to target DNA to particular cell types (Machy et al. (1988) Proc. Natl. Acad. Sci. USA 85:8027-8031; Trubetskoy et al. (1992) Bioconjugate Chem. 3:323-27 and WO 91/17773 and WO 92/19287).
Peptides derived from the amino acid sequences of viral envelope proteins have been used in gene transfer when coadministered with polylysine DNA complexes (Plank et al. (1994) J. Biol. Chem. 269:12918-24). Trubetskoy et al. (supra) and Mack et al. ((1994) Am. J. Med. Sci. 307: 138-143) suggest that cocondensation of polylysine conjugates with cationic lipids can lead to improvement in gene transfer efficiency. WO 95/02698 discloses the use of viral components to attempt to increase the efficiency of cationic lipid gene transfer.
Disulfide bonds have been used to link the peptidic components of a delivery vehicle (Cotten et al. (1992) Meth. Enzymol. 217:618-644); see also, Trubetskoy et al. (supra) However, the chemical modification of the various components, although group specific, is not regio-specific and leads to enormous molecular heterogeneity of the conjugated product. Disulfide bonds are also known to be unstable in biological fluids and thus limits the potency of such compounds in practice.
Similar heterogeneity is also produced by other standard conjugation methods such as carbodiimide coupling through side chain carboxyl groups (Wu et al. (1991) J. Biol. Chem. 266:14338-42). However, in addition to the above disadvantages, the resulting amide bond coupling the components is chemically stable within the cytosol and makes the components difficult to separate.
More specific coupling chemistry has been employed by Cotten et al. (supra). This method involves oxidation of the carbohydrate moieties using periodate, followed by subsequent reaction with polylysine. The Schiff base so formed was reduced with sodium cyanoborohydride to form a stable amide bond. However, due to the large number of available lysine residues, the resulting amide bond was linked at random to the polylysine component.
Trubetskoy (supra) observed increased efficiency of a conjugate made up of a heterogeneous polylysine moiety linked through the N-terminus non-specifically to amino functions on a monoclonal antibody.
Many prior art methods employ highly heterogeneous components linked by conjugation chemistry which itself leads to more heterogeneity. This heterogeneity leads to poor control during preparation and large batch-to-batch variability, low potency and poor solution stability.
Scale up and reproducible manufacture of the gene delivery vehicles described in the literature are problematic because of the extreme heterogeneity of the products and components of those systems. Key parameters such as quality control, process control and product identification are thus rendered imprecise. Therefore, an object of the invention is the development of a reproducible and scalable production process for pharmaceutical compositions which facilitate delivery of exogenous DNA to a target cell with high efficiency.
Another object of the invention is to provide an improved transfection complex having chemical components of defined stoichiometry and therefore reduced heterogeneity.
Yet another object of the invention is to provide pharmaceutical formulations for transfection which exhibit increased transfection efficiency.
The invention is based on the discovery of polypeptides which, when associated with a nucleic acid, confer a high efficiency upon host cell transfection.
The invention encompasses a polypeptide comprising or consisting of the following 29 amino acid composition: 6G""s, 2F""s, 2L""s, 1W, 4R""s, 2E""s, 2N""s, 3K""s, 1T, 1S, 1A, 1Y, 1M, 1C, and 1I, and having additional cationic residues to provide a net number of positive charges of greater than 8. Preferably, the 29 amino acids are contiguous.
As used herein, xe2x80x9ccompositionxe2x80x9d refers to the amino acid content rather than an order of amino acids. xe2x80x9cCationic residuexe2x80x9d refers to an amino acid or other molecule having a net positive charge, examples of which include but are not limited to lysine, ethyleneimine, arginine, methacrylate, amidoamine, protamine, spermine, and spermidine. xe2x80x9cCationic chargexe2x80x9d refers to a net positive charge.
As the 29 amino acids specified above contained in the polypeptide contain 7 cationic residues and 2 anionic residues, and thus contain a net of 5 cationic charges, the additional cationic charges will number at least 3, preferably, 4 or 5 and more preferably number, for example, 6, 12, 18 or 24.
Polypeptides according to the invention will therefore contain the 29 amino acids specified above and additional cationic residues sufficient to net equal to or greater than 8 positively charged residues in the polypeptide, wherein the 29 amino acids specified above may be present in the polypeptide as (a) a block of: 29 contiguous amino acids and equal to or greater than (xe2x89xa7) 3 cationic residues (monomer) or (b) as two or more blocks of the 29+xe2x89xa73 amino acids (dimer, trimer, etc., i.e., multimer). Where a 29+xe2x89xa73 polypeptide is present in a multimeric form, several individual 29+xe2x89xa73 polypeptides may be linked in conventional stable bonds (peptide, oxime, or thioether) or the polypeptides may be linked via labile bonds, e.g., disulfide bonds.
A cationic sequence useful herein may be a tract of contiguous cationic residues in the length range of 3 to 700, whereby the net cationic charge is at least 3. Alternatively, the tract of cationic sequences need not be contiguous, but may be dispersed among basic or neutral amino acids such that the net number of cationic charges is in the range of 3 to 700. Therefore, a polypeptide according to the invention may contain as few as (5+3=)8 net cationic charges or as many as (5+700=)705 net cationic charges. A given cationic sequence may be linked in conventional stable bonds at either the amino or carboxy terminus of the 29 amino acid tract specified above, or at both ends, or within the 29 amino acid tract. If cationic sequences are present at both ends of the 29 amino acid tract, then each end may contain a different number of cationic residues or an identical number of cationic residues. Alternatively, the cationic sequence may be bonded to the 29 amino acid tract at one or more position along its length via a labile (sulfhydril) or a stable bond. Finally, the 29 amino acid tract may be linked to a cationic sequence at one or more position along the length of the cationic sequence via a labile or stable bond.
The invention also encompasses a polypeptide comprising the amino acid sequence, from amino to carboxy termini,
NH2-KKKKKKGGFLGFWRGENGRKTRSAYERMCNILKGK-COOH (SEQ ID NO:1) (also referred to herein as K6CL22, K6CLII, CL22 or CLII).
The invention also encompasses a polypeptide comprising the amino acid sequence, from amino to carboxy termini, 
Preferably, the above-polypeptides consists essentially of the above-recited sequences.
The invention also encompasses a transfection complex comprising a polypeptide comprising the amino acid sequence, from amino to carboxy termini,
NH2-KKKKKKGGFLGFWRGENGRKTRSAYERMCNILKGK-COOH (SEQ ID NO:1), and an isolated nucleic acid.
The invention also encompasses a transfection complex comprising a polypeptide comprising the amino acid sequence, from amino to carboxy termini, 
The invention also encompasses a dimerized K6CL22 polypeptide comprising the amino acid sequence, from amino to carboxy termini, 
wherein Sxe2x80x94S refers to a cystine linkage (disulfide bond) between each cysteine residue of the two peptides.
The invention also encompasses a polypeptide comprising the following amino acid sequence from amino to carboxy terminus:
H-KKKKKKGGFLGFNTKERNLKRGWEICRSAMGYGRK-OH (SEQ ID NO:2) (CL28).
The invention also encompasses a polypeptide comprising the following amino acid sequence from amino to carboxy terminus:
H-KKKKKKKKKKKKGGFLGFWRGENGRKTRSAYERMCNILKGK-OH (SEQ ID NO:3) (CL26).
The invention also encompasses a dimerized CL26 polypeptide comprising the following amino acid sequence from amino to carboxy terminus: 
wherein Sxe2x80x94S refers to a disulfide bond between each cysteine residue of the two polypeptides.
The invention also encompasses the polypeptide referred to herein as NBC30, comprising the following: 
wherein Sxe2x80x94S refers to a disulfide bond between each cysteine residue of the two polypeptides.
As used herein, the term xe2x80x9ctransfection complexxe2x80x9d refers to a mixture of a peptide according to the invention and a nucleic acid which is preferably DNA but which also may be RNA, the nucleic acid of which is condensed.
The invention also encompasses a transfection complex comprising a mixture of polypeptides of two or more different amino acid sequences, wherein the mixture of polypeptides includes as one of the polypeptides of the mixture any one of the polypeptides (monomers or multimers) described above. For example, one polypeptide of the mixture is the polypeptide having the sequence
NH2-KKKKKKGGFLGFWRGENGRKTRSAYERMCNILKGK-COOH (SEQ ID NO: 1), and an isolated nucleic acid.
Another example of a transfection complex according to the invention comprising a mixture of polypeptides of two or more different amino acid sequences, wherein the mixture of polypeptides includes as one of the polypeptides of the mixture the polypeptide having the sequence 
As used herein, the phrase xe2x80x9cdifferent amino acid sequencesxe2x80x9d refers to a sequence that differs from one of the above described amino acid sequences at one or more residues, or that differs from this sequence in length by one or more residues.
It is preferred that the polypeptide and nucleic acid are associated such that said nucleic acid is condensed.
The invention also encompasses a pharmaceutical formulation for taansfection of cells, comprising an amino acid sequence as described above, an isolated nucleic acid, and a pharmaceutically acceptable diluent.
Preferably, the polypeptide and said nucleic acid are associated such that said nucleic acid is condensed.
The invention also encompasses host cells containing the polypeptide or the transfection complex described herein, and methods of transfection.
The transfection methods encompassed by the invention include wherein a host cell is contacted with the transfection complex, or the pharmaceutical formulation containing the transfection complex.
Although the host cell may be any type or species of cell, it is preferred that the host cell is a eukaryotic cell, such as a mammalian cell, including cells that are human, mouse, monkey, hamster, and the like. Cell types transfectable according to the invention include somatic cells, including primary cells as well as cell lines, such as dendritic cells, tumor cells, fibroblasts, muscle cells, and germline cells, such as ovarian cells.
The methods also include introducing a nucleic acid into a host cell in vivo by administering to a patient a transfection complex or a pharmaceutical formulation according to the invention.
The methods also contemplate improvements over known methods of delivering a nucleic acid to a cell wherein a nucleic acid delivery complex is administered to a patient, the improvement comprising wherein the delivery complex comprises a nucleic acid and a polypeptide mixture which includes as one of the polypeptides of the mixture the polypeptide having the composition or sequences described herein.
The invention also contemplates a method of preparing a transfection complex, comprising contacting the a polypeptide according to the invention, or a mixture of polypeptides containing this polypeptide, with a nucleic acid under conditions which permit the condensation of the nucleic acid and the association of the polypeptide and the condensed nucleic acid in a particle.